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GTP‐induced fusion of isolated pancreatic microsomal vesicles is increased by acidification of the vesicle lumen
Author(s) -
Hampe Wolfgang,
Zimmermann Petra,
Schulz Irene
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80372-p
Subject(s) - gtp' , vesicle , guanosine , chemistry , guanosine triphosphate , biochemistry , atpase , biophysics , biology , membrane , enzyme
Using the ‘fusogen’ polycthyleneglycol (PEG), Dawson et al. [1] have concluded that both guanosine triphosphate (GTP)‐mduced calcium efflux and the enhancement of IP 3 ‐promoted calcium release from rat liver microsomal vesicles could be attributed to a GTP‐dependent vesicle fusion. We have studied GTP‐induced fusion of microsomal vesicles from rat exocrine pancreas using light scatter and fluorescence dequenching methods. In the presence of PEG (3%), GTP (10 μM) induced a decrease in light scatter and an increase in fluorescence in the fluorescence dequenching assay (GTP‐effect) indicating fusion of the vesicles. Guanosine 5'‐ O ‐(3‐thiotriphosphate) (10 μM) had no effect on its own and inhibited the GTP‐induced signals. Preincubation of the vesicles with adenosine triphosphate (ATP) (4 mM) increased the GTP‐effect by 80%. whereas bafilomycin B 1 , a specific inhibitor of vacuolar type H + ‐ATPases, and the protonophore CCCP (10 μM) inhibited only the ATP‐dependent part of the GTP‐effect. Inhibitors of the vacuolar type H + ‐ATPase, which are also SH‐alkylating reagents such as N ‐ethylmaleimid (100 μM) and the tyrosine‐,cysteinc‐ and lysine‐reactive reagent 7‐chloro‐4‐nitrobenz‐2‐exa‐1,3‐diazole (10 μM) [2,3j. abolished the GTP‐effect in the absence or presence of ATP. We conclude that GTP induces fusion of pancreatic microsomes which is increased by an H + gradient established by a vacuolar type H + ‐ATPase.